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Mouse Surgery Protocol ( Click to Download ) Mouse Surgery Protocols (edited 1/11/96) A. 2,2,2-tribromoethanol (Avertin™) — From Papaioannou and Fox (1993); ref#2008 Solvents for 2,2,2-tribromoethanol: Add 2.5 g tribromoethanol (Aldrich) to 5 ml 2-methyl-2-butanol (tertiary amyl alcohol; Aldrich) and dissolve by heating to 50oC with stirring or shaking. Add 200 ml distilled water and […]
Mouse Surgery Protocol ( Click to Download ) A. Transplantation of mammary epithelium into cleared fat pads Prepare the following equipment: Cautery and spare batteries Surgical tools: DeWecker iris scissors (7mm, sharp-sharp); 2 curved iris forceps; tissue forceps; needle-point forceps; hemostat; sterile sutures Ear tags Auto-wound clips and remover too Avertin on ice 25 gauge […]
Freeze Media 9ml media the cells grow in 1ml DMSO Trypsinize as normally. Spin cells and resuspend in appropriate freeze media for ~1-2million cells/vial. Place 1ml of Freeze Media. Place cryovial in Nalgene cell freezer and put at -80°C until next day. (If you do not use a Nalgene cell freezer, this protocol will NOT […]
Collecting CM From CAFs or RMFs ( Click to Download ) Seed fibroblasts at one of the following cell densities depending on cell number: 100% confluence, 106 cells per 10 (dense) or 15 cm (sparse) plate, or 5×106 cells per 15 cm plate. Be consistent if comparing CM potencies among various patient fibroblasts. Try to […]
Crystal Violet Staining Protocol for Quantifying Proliferation Plate 25k cells in a 12 well plate or 50k in a 6-well plate Keep an empty well w/media to use as a blank Aspirate media, wash cells 1x with PBS Fix the cells with 10% buffered formalin for 30 min (use 1mL/well) Wash the cells 1x with […]
Culturing HMECs ( Click to Download ) HME/C lines are slower to trypsinize than most cells. Use care when passaging to avoid over-trypsinizing and damaging cells. Grow cells to 70-80% confluence in MEGM media. Aspirate media from the plate; rinse with 10 ml PBS to remove cellular debris and traces of serum (though most […]